Three types of transmembrane proteins are expressed in the membrane of influenza type A virions and virus-infected cells. The hemagglutinin and neuraminidase are glycoproteins with large ectodomains of ˜510 and ˜420 amino acids, respectively. Hemagglutinin is assembled as homotrimers and neuraminidase as homotetramers forming a dense layer of 13-14 nm long, rod-shaped surface projections on the viral membrane and at cellular sites of virus maturation. Current influenza virus vaccines aim at inducing a strong antibody response to these glycoproteins, particularly the hemagglutinin, as such antibodies are well-known to be highly protective against infection. The problem is that influenza type A virus has a high propensity for changing the determinants recognized by these protective antibodies, which necessitates repetitive vaccination with updated vaccine strains that reflect these antigenic changes. By contrast, the third viral transmembrane protein, matrix protein 2 (M2), contains an ectodomain (M2e) that is highly conserved amongst human influenza virus strains. Broad protective immunity against influenza type A virus infection using M2 has been investigated (Slepushkin, et al. (1995) Vaccine 13:1399-1402; Frace, et al. (1999) Vaccine 17:2237-44; Neirynck, et al. (1999) Nature Med. 5:1157-63; Okuda, et al. (2001) Vaccine 19:3681-91).
M2 is a 97 amino acid non-glycosylated transmembrane protein (Lamb, et al. (1981) Proc. Natl. Acad. Sci. USA 78:4170-4; Lamb, et al. (1985) Cell 40:627-33). It forms homotetramers (Holsinger and Lamb (1991) Virology 183:32-43; Sugrue and Hay (1991) Virology 180:617-24) that are expressed at low density in the membrane of virus particles (˜10 M2 tetramers compared to ˜400 hemagglutinin trimers and ˜100 neuraminidase tetramers per average virion) but at high density in the plasma membrane of infected cells (similar density as hemagglutinin) (Zebedee and Lamb (1988) J. Virol. 62:2762-72). M2-tetramers exhibit pH-inducible proton-transport activity (Steinhauer, et al. (1991) Proc. Natl. Acad. Sci. USA 88:11525-9; Pinto, et al. (1992) Cell 69:517-28) which appears to facilitate the release of RNP complexes from the viral membrane after fusion (Zhirnov (1990) Virology 176:274-9) and prevents an excessive drop of pH within transport vesicles during egress of viral transmembrane proteins from endoplasmic reticulum to the plasma membrane, thereby preventing a premature acid-induced conformational change in hemagglutinin (Steinhauer, et al. (1991) supra). The 23 amino acid long M2e is totally conserved in its nine N-terminal amino acids and shows only a relatively minor degree of structural diversity in its membrane-proximal 15 amino acid long section (Zebedee and Lamb (1988) supra; Ito, et al. (1991) J. Virol. 65:5491-8). Amongst human isolates of H1N1, H2N2, H3N2, and H5N1 subtypes, two alternative amino acids have been found at seven positions but the majority of human isolates actually share the same sequence.
M2e-specific monoclonal antibody 14C2 does not prevent virus infection in vitro but reduces virus yield and plaque size when incorporated into the culture medium or agar overlay (Zebedee and Lamb (1988) supra; Hughey, et al. (1995) Virology 212:411-21). Not all M2e-specific antibodies display this activity (Hughey, et al. (1995) supra) and not all virus strains are susceptible to it (Zebedee and Lamb (1988) supra). In vivo, passive monoclonal antibody 14C2 similarly decreases virus growth (Treanor, et al. (1990) J. Virol. 64:1375-7) and is effective also against PR8 (Mozdzanowska, et al. (1999) Virology 254:138-46), which is not susceptible to antibody-mediated growth restriction in vitro (Zebedee and Lamb (1988) supra; Mozdzanowska, et al. (1999) supra), indicating that antibody-mediated virus growth-inhibition occurs through distinct mechanisms in vitro and in vivo.
The protective efficacy of actively induced M2-specific immunity has been tested using various types of vaccine constructs and vaccination modalities. Initial studies, in which mice and ferrets were vaccinated with M2-expressing, recombinant vaccinia virus, showed no evidence of protection (Epstein, et al. (1993) J. Immunol. 150:5484-93; Jakeman, et al. (1989) J. Gen. Virol. 70:1523-31), although the induction of M2-specific immune responses was not verified. Subsequent studies tested plasmid DNA containing the intact M gene segment (coding for M1 and M2 protein) (Okuda, et al. (2001) supra), an intact recombinant M2 protein membrane preparation (Slepushkin, et al. (1995) supra), an M2 protein with a deleted transmembrane portion (to decrease toxicity and increase solubility) (Frace, et al. (1999) supra), and a construct in which M2e was fused to hepatitis B virus core protein (Neirynck, et al. (1999) supra). These latter vaccination protocols induced protection, both in terms of reduction in virus growth and mortality.
It has now been found that a multiple antigenic agent containing M2e linked to helper T cell determinants is an effective vaccine for inducing virus protection. M2e-MAAs together with cholera toxin (CT) and a synthetic oligodeoxynucleotide (ODN) with a stimulatory CpG motif induces strong M2e-specific antibody titers in serum of mice and results in significant protection against influenza virus challenge.